Method and apparatus for thin layer chromatography

ABSTRACT

METHOD AND APPARATUS ARE PROVIDED FOR CARRYING OUT CHROMATOGRAPHS ON A THIN LAYER OF ADSORBENT, WHERE THE THIN LAYER OF ADSOREBET HAS A PLURALITY OF HOLLOWS ADJACENT TO ONE END. INSERTED IN AT LEAST ONE OF THE HOLLOWS IN AN INSERT WHICH IS IMPREGNATED WITH AT LEAST ONE COMPOUND OF KNOWN CHEMICAL COMPOSITION AND INSERTED IN AT LEAST ONE OF THE OTHER HOLLOWS IS AN INSERT IMPREGNATED WITH A SOLUTION HAVING ONE OR MORE UNKNOWN COMPOUNDS. THE DEVELOPMENT OF THE CHROMATOGRAPH IS THEN CARRIED OUT IN A NORMAL MANNER. BY COMPARING THE DISTANCE THE UNKNOWN COMPOUND HAS TRAVELED WITH THE DISTANCE THE KNOWN COMPOUND OR COMPOUNDS HAVE TRAVELED AND THKEIR RESPONSE TO VARIOUS DEVELOPING MATERIALS, SUCH AS DYES, OXIDANTS, ETC., THE UNKNOWN COMPOUND CAN BE DETERMINED, AS WELL AS A QUALITATIVE ESTIMATE OF THE AMOUNT.

Jan. 30, 1973 D. w. JONES 3,714,035

METHOD AND APPARATUS FOR THIN LAYER CHROMATOGRAPHY Filed May 19, 1971 |s/'O I 1 Fl G 2 FIG .3 I DONALD W S R J ES ATTORNEYS US. Cl. 210-31 C 22Claims ABSTRACT OF THE DISCLOSURE Method and apparatus are provided forcarrying out chromatographs on a thin layer of adsorbent, where the thinlayer of adsorbent has a plurality of hollows adjacent to one end.Inserted in at least one of the hollows is an insert which isimpregnated with at least one compound of known chemical composition andinserted in at least one of the other hollows is an insert impregnatedwith a solution having one or more unknown compounds. The development ofthe chromatograph is then carried out in a normal manner. By comparingthe distance the unknown compound has traveled with the distance theknown compound or compounds have traveled and their response to variousdeveloping materials, such as dyes, oxidants, etc., the unknown compoundcan be determined, as Well as a qualitative estimate of the amount.

BACKGROUND OF THE INVENTION Field of the invention Thin layerchromatography (TLC) is an extremely versatile and economical method forseparation analysis of unknown compositions. Extremely simple equipmentis employed, and relatively simple manipulation and handling isrequired. Thin layer chromatography has found widespread use in thebiomedical field for analyzing for drugs, e.g. narcotics, tranquilizers,barbiturates, amphetamines, etc., naturally occurring biologicallyactive materials, e.g. hormones, prostaglandins, steroids, amino acidsand polypeptides, as well as other materials, such as pesticides,organic toxins, etc. Thin layer chromatography requires extremely smallamounts of sample, usually in the order of a fraction or a fewmicrograms, so that it is ideally suited for situations where extremelylow concentrations of materials are encountered.

Because of the convenience of thin layer chromatography, an extensiveliterature has developed detailing the use of solvents, adsorbents anddeveloping agents in the analysis of a wide variety of compounds. The Rvalues have been reported for a wide range of compounds under diverseconditions. (R, is the ratio of the distance the compound travels to thedistance the solvent boundary travels.) Nevertheless, this value issensitive to variations in conditions and it is usually desirable tohave a standard, so as to provide a direct comparison. While someinformation about the nature of the unknown material may be determinedby the method of isolation, there frequently remains a wide range ofpossibilities as to the chemical composition of the unknown. Therefore,it has been found necessary in most instances to maintain a relativelylarge number of standard solutions with which to compare the unknown tothe standard.

In addition, in preparing the chromatograph, it is desirable to have arelatively discrete spot of the unknown solution. The larger or morediffuse the spot, which is initially introduced onto the chromatograph,the larger and more diffuse the spot becomes as it moves along thechromatograph. This has required relatively painstaking eiiorts ofslowly introducing minute quantities of the un- United States Patent Oknown solution onto the chromatograph, drying the spot, and then addinganother minute quantity, until the total amount of unknown material hasbeen added.

Description of the prior art For a general discussion of thin layerchromatography see Stahl, Thin Layer Chromatography, Springer-Verlag,New York, 1969; and Thin Layer Chromatography Technical Bulletin No. 22Brinkmann Instruments, Inc., New York 1962.

SUMMARY OF THE INVENTION Thin layer chromatographs are scored, so thatby removal of the scored area, hollows are provided into which insertsmay be introduced. The scored areas are adjacent one end of thechromatograph. Inserts are provided which are impregnated with one ormore standardized compositions and which may be inserted snugly into thescored area. In addition, blank inserts are provided for impregnationwith unknown solution which maybe introduced into one or more of thescored areas. The inserts fit snugly into the scored area so as toprovide a substantially con tinuous surface. The chromatograph is thendeveloped in the normal way by allowing the solvent system to migratefrom one end of the chromatograph toward the other end for a substantialdistance. The spots are then developed according to known means. Bycomparison of the standard with the unknown, the presence of any one ofthe standard compounds in the unknown may be determined.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view of a thinlayer chromatop FIG. 2 is a side elevational view of a thin layerchromatograph with a backing; and

FIG. 3 is a perspective view of a disc.

DESCRIPTION OF THE SPEOIFIC EMBODIMENTS A variety of thin layerchromatographs are available which employ different absorbents andditferent means for stabilizing the adsorbents. Usually, the thicknessof the adsorbent layer will be from about 0.2 to about 2 millimeters,more usually from about 0.5 to about 1.5 millimeters. Adsorbents whichfind widespread use are silica gel, alumina, kieselguhr, carbon black,magnesium oxide, etc. The chromatographs may be stabilized in a varietyof ways, by adhering the adsorbent to a backing, such as foil or glass,or by impregnating a web of glass fiber, the latter being preferred forthis invention.

The usual thin layer chromatograph will be about 1 to 10 cm. in width,and from about 10 to cm. in length. Of course, any width or length maybe used in this invention, depending on the size of the equipmentavailable, the number of materials involved, as well as the availabilityof such thin layer chromatographs.

In carrying out this invention, a conventional thin layer chromatographcan be employed and at a distance from about 1 to 3 centimeters, moreusually about 2 centimeters, from one end, a number of small areas arescored, so as to allow for easy removal of the scored area to provide ahollow area. These scored areas may be removed at the time offabrication of the thin layer chromatograph or may be supplied aspresent in the thin layer chromatograph, prior to use by thechromatographer. To the extent a plurality of areas are scored, it willbe more convenient to leave the scored areas present, so that only thatnumber which is required may be removed to provide a hollow forinsertion of an insert.

Standard inserts are provided which are impregnated with one or morestandard compositions at known concentrations. One or more of theinserts impregnated with the same or different standards are pressedfirmly into the hollows or openings so as to provide a substantiallycontinuous surface for the chromatograph. One or more inserts areimpregnated individually with one or more of the same or differentunknowns and similarly inserted into available hollows.

The normal chromatography procedure is then followed, by introducing thechromatograph into a jar having sufiicient solvent, so that thechromatograph is immersed to the extent of a few millimeters. Thesolvent is allowed to migrate, normally to a fixed distance from theend, at which time the chromatograph is removed from the jar and dried.

Depending on the compounds involved, the compounds may be observed forfluorescence, or developed with dyes, oxidants, etc. Chromatographicadsorbents frequently have materials incorporated which permit directobservation of spots with certain compounds. For example, fluorescingmaterials may be incorporated and compounds which quench fluorescencewould be observable. By comparison of the spot formed by the unknownwith the spots from the standards, a determination may be made if one ormore of the standard materials was present in the original unknown andqualitatively the amount of the material.

The inserts which are provided may be of any shape which conforms withthe scored area on the chromatograph. Most conveniently, the scored areawill be circular, but may be rectangular, square or other shape. Thesize of the scored area will normally have as its largest dimensionapproximately A; inch to inch, preferably about 3 inch, Blank insertsare provided which are used for the unknown, by dipping the insert intoa solution containing the unknown and then drying the insert. Theinserts may be made of any appropriate material, such as supportedadsorbent (see subsequent discussion), filter paper or other adsorbentmaterial.

The standards are conveniently prepared by spraying a sheet of theadsorbent material, drying the sheet and cutting out the inserts.

The standard inserts may have more than one component, particularlywhere there are a family of compounds which have similar properties andare separated by the same adsorbent and solvent system. Desirablecombinations would be morphine and morphine analogs; barbiturates,certain groups of tranquilizers, e.g. benzdiazacycloheptanes; variousgroups of steroids; prostaglandins; popular phosphate insecticides;popular carbamate insecticides; halogenated fungicides; halogenatedinsecticides; amphetamines, etc. Depending upon the ease of detection,as little as one microgram of the unknown compound need be present andalmost never more than one milligram, more usually in the range of about2 micrograms to 500 micrograms and preferably in the range of about 2micrograms to 50 micrograms. The amount of material impregnated in theinsert can be easily determined by the amount of solution of knownconcentration adsorbed by the insert.

The following are illustrative families of materials which may beimpregnated in a single disc:

Morphine and related compounds normally have a tertiary amine and one ormore oxy or x0 functionalities. Illustrative morphine and physiomimeticanalogs are hydromorphone, oxymorphone, codeine, hydrocodone,pholcodine, dextromethorphan, phenazocine and dionin.

Physiomimetic analogs to morphine include methadone and structuralanalogs, illustrated by dextromoramide, dipipanone, phenadoxone,propoxyphene (Darvon) and acetylmethadone.

Another class of compounds includes meperidine and its analogs, such asalphaprodine, alvodine and anileridine.

Another class of compounds are the catecholamines, illustrated bycotainine, narceine, noscapine, papaverine, epinephrine, and L-dopa.Analogous compounds of catecholamines are those having unhydroxylatedphenyl rings which include amphetamine, paredrine, ephedrine,methamphetamine and norephedrine.

Another class of compounds are the barbiturates which are5,5-disubstituted barbituric acids or their sodium salts. Thesubstituents at the 5 position are normally hydrocarbon, usually from 1to 6 carbon atoms. Illustrative barbiturates are veronal, medinal,luminal, prominal, soneryl, nembutal, amytal, dial, phenadorn, seconal,evipan, phenobarbital and pentothal.

Another drug of importance is cocaine and its hydrolytic productecgonine.

Among the steroidal families of interest are the androgens (malehormones), estrogens (female hormones), gestogens and corticosteroids,including the mineralcorticolds and glucocorticoids.

Another compound of interest is tetrahydrocannibinol which is an activeingredient of marijuana.

Another group of compounds which are of interest are tranquilizers,which include Meprobamate, benzdiazocycloheptanes, such as Librium,Diazepam or Oxazepam, and phenothiazin such as Chlorpromazine, Vesprin,Phenergan, Diparcol, and Pyrrolazote.

Another group of compounds are the sapogenins and saponins, of whichdigitalis finds widespread use.

Other groups of compounds include the vitamins, which because of theirwidely different structure, would probably be analyzed only for membershaving similar chemical structure, such as the D vitamins. Also, varioussaccharides and polysaccharides may be analyzed. As already indicated,pesticides can be analyzed, normally having such groups as phosphates,carbamates, thiocarbamates, sulfenamides, halogenated polycyclics, etc.

Also, various polypeptides and amino acids may be analyzed, such astryptophan, alanine, glutamine, aspartic acid, tyrosine, cysteine,angiotensin, luteinizing hormone, insulin, follicle stimulating hormone,etc.

Of course, the above list is intended only to be illustrative, since thesubject invention may be employed with any material which can bechromatographed, providing a convenience both in preparing the spot forthe chromatograph and the standard for comparison with the unknown.

The method of this invention will now be indicated with a specificexample with consideration of the draw ings. First, an unknown sample isobtained, either from such biological fluid as urine, saliva, blood, orother fluid, and treated in an appropriate manner, so as to permitextraction of a class of compounds. For example, if basic compounds suchas amines were to be extracted, normally a high pH would be used. Ifacid compound were to be extracted, normally a low pH would be used. (Ofcourse, the subject invention may be used with other than body fluids,such as scrapings from plants, digested plants, pollutants, or anymaterial which has a component which is to be assayed.)

After treating the biological fluid in an appropriate way, the solutionis then extracted with a water immiscible solvent so as to provide anorganic and an aqueous layer. The organic layer should have the unknownmaterial to be assayed.

Depending on the particular assay, the organic solution may then betreated in a variety of ways to remove possible interferants, tostabilize the solution, or even to modify the unknown, so as to make itmore amenable to chromatography. After the appropriate treatment, theorganic solution is then concentrated to the desired concentration, soas to provide suflicient material on the insert.

In accordance with this invention, a large number of inserts 10 areprovided, as depicted in FIG. 3, which are most conveniently circular.The chromatograph 12 is usually rectangular having a locating marker 14at one end. If desired, a boundary line 16 is drawn which indicates thedistance to which the boundary of the solvent is to be allowed to go.

A plurality of scored areas 18 are provided which are adjacent to theedge opposite the locating marker 14. Two or more scored areas areprovided, which are scored so as to allow for the easy removal of thescored portion to leave a hollow area. The insert is shaped the same asthe scored area and fits snugly therein, providing a substantiallycontinuous surface through which the solvent migrates.

Two or more of the scored areas 18 are removed so as to provide placesfor the introduction of inserts. A blank insert 10 is taken and dippedinto the concentrated unknown solution absorbing the unknown solution.The insert is then allowed to dry and inserted into one of the hollowareas formed by removal of the scored portion. Preferably, although notessential, the unknown is placed in the middle. Assuming one isinterested in a particular class of compounds, or a number of differentclasses of compounds which can be chromatographed by the particularabsorbent and solvent system employed, one or more standards may beinserted into one or more of the hollow areas which are provided.

The inserts are self-supporting so that means must be provided for theirnot crumbling under normal handling conditions. This can be achieved ina variety of ways. With chromatographys which have a backing 20 such asglass or a foil, supporting an adsorbent layer 21, the insert may alsobe made of an adsorbent which is adhered to a backing, Where the backingis glass, it will normally be preferable to provide the necessarystability using glass fiber, such as employed in conventional,commerically available thin layer chromatographs. Other layers may alsobe provided for stabilizing the insert. It is found, that the insertwhich is supported by glass fiber can be used as an insert in the otherchromatographs, such as the foil and glass backed chromatographs.Alternatively, various grades of filter paper may be employedadvantageously.

After inserting the desired number of inserts with the appropriatestandards and unknows, the chromatograph 12 is introduced into a jarwhich has a sufiicient amount of the solvent system to immerse thebottom of the chromatograph a few millimeters. A cover is put over thejar to prevent evaporation, and the solvent is allowed to migrate untilit reaches the boundary marker 16. This is usually a factor of fifteenminutes to a few hours. The chromatograph is then removed from the jar,the solvent allowed to evaporate and then, depending upon the particularcompounds to be observed, the chromatograph developed in a number ofways.

As already indicated, a number of compounds respond in specific ways tocertain dyes. Alternatively, the chromatography may be introduced into ajar of iodine vapor and developed. In addition, certain materials aresensitive to oxidants and either decolorize the colored oxidant, orbecome colored so as to be identifiable. By comparison of the color ofthe standard, in relation to the unknown,

a qualitative determination of the amount of unknown may be made.

In FIG. 1, the chromatograph is indicated after developing, where twounknowns were employed, and a single standard having four difierentconstituents. The chromatograph would indicate that the second sampledoes have one of the components present in the spots provided by thestandard 24. The other unknown would have a component 26 which is notpresent in the standard.

In accordance with this invention, a simplified method is providedwhereby a large stock pile of standards can be conveniently maintainedfor use in thin layer chromatography. In this manner, thin layerchromatographic analysis can be greatly simplified by direct comparisonof the standard to the unknown on the same chromatograph. In addition,applying the unknown to the chromatograph is greatly simplifiedproviding a simpler more efficient way of insuring localized applicationof the unknown to the chromatograph.

The subject invention is extremely advantageous in analyticallaboratories. The number of even common drugs, which are subject toabuse and over-dosage, is great. To have smaller laboratories obtain alibrary of pure drugs in the proper form is presumptuous. Even largelaboratories with unlimited facilities are deficient in not havingenough standard drugs to fully identify recovered spots in unknowns.Pure compounds are generally unavailable through chemical supply housesand frequently must be obtained from manufacturers. When one considersthe large number of drug manufactures, some idea is obtained as to thedifliculty of forming a drug library. The problem is enhanced since theuse of pills or capsules to obtain standard drug material is frequentlyunsatisfactory. Additionally, because of the abuse potential andassociated stringent laws, many of the drugs, e.g. morphine and heroin,are obtained only after great difficulty. The subject invention obviatesthese problems and provides an efiicient and convenient library of drugsfor thin layer chromatographic analysis.

What is claimed is:

1. Method for chromatographing an unknown solution employing a thinlayer chromatograph having an adsorbent surface with a small hollow areaadjacent one end of said chromatograph which comprises:

immersing in said solution at the desired concentration,

a self supporting insert, which is shaped to fit snugly in said hollowarea;

inserting the insert into said hollow; and

chromatographing with a solvent system, wherein the solvent systemmigrates from the boundary adjacent said hollow toward the opposite endof said thin layer chromatograph.

2. A method according to claim 1, wherein said insert is dried afterimmersing and prior to inserting into said hollow.

3. A method according to claim 1, wherein said adsorbent surface iscomprised of silica gel or alumina and is supported by glass fiber,glass or foil.

4. A method according to claim 1, wherein said insert is circular and isof from A3" to A" in diameter.

5. A method according to claim 1, wherein a plurality of hollow areasare provided which are equidistant from one end of said thin layerchromatograph, and wherein in at least one hollow area, an insertimpregnated with at least one compound of known composition is insertedprior to chromatographing.

6. A method according to claim 5, wherein said compound of knowncomposition is a biologically active compound.

7. A method according to claim 1, wherein said insert is impregnatedwith a plurality of compounds of known composition belonging to theclass of narcotics, barbiturates, amphetamines, steroids,catecholamines, or tranquilizers.

8. A thin layer chromatograph having a plurality of scored or hollowareas, the hollow areas obtained by removal of the scored portion;

the scored or hollow area being in a linear arrangement paralleling andadjacent one end of said thin layer chromatograph. v

9. A thin layer chromatograph according to claim 8, wherein theadsorbent of said chromatograph is silica gel or alumina and saidadsorbent is supported by glass fiber or foil.

10. Self-sustaining adsorbent inserts usable in a method according toclaim 1;

which are of substantially regular shape, having a selfsustainingadsorbent layer; and

their largest dimension in the range of A3" to A".

11. Inserts according to claim 10, wherein said insert is composed ofsilica gel or alumina supported by glass fiber or foil.

12. Insert according to claim 11, wherein said insert is composed ofsilica gel supported by glass fiber.

13. Insert according to claim 10, impregnated with at least one memberof the class of barbiturates in an amount of about 1 to 500 micrograms.

14. Insert according to claim 10, impregnated with at least one memberof the class of narcotics in an amount of about 1 to 500 micrograms.

15. Insert according to claim 10, impregnated with at least one memberof the class of tranquilizers in an amount of about 1 to 500 micrograms.

16. Insert according to claim 10, impregnated with at least one memberof the class of steroids in an amount of about 1 to 500 micrograms.

17. Insert according to claim 10, impregnated with at least one memberof the class of amphetamines in an amount of about 1 to 500 micrograms.

18. Insert according to claim 10. impregnated with at least one memberof the class of catecholamines in an amount of about 1 to 500micrograms.

19. Insert according to claim 10, impregnated with at least one memberof the class of polypeptides in an amount of about 1 to 500 micrograms.

20. Insert according to claim 10, wherein said selfsustaining adsorbentlayer is filter paper.

21. Insert according to claim 10, impregnated with at least one memberof the class of hypnotics in an amount of about 1-500 micrograms.

22. Insert according to claim 10, impregnated with at least one memberof the class of central nervous system stimulants, in an amount of about1-500 micrograms.

References Cited UNITED STATES PATENTS 3,046,779 7/1964 Coleman 21031 C3,318,451 5/ 1967 Przrbrlowicz 210-31 C 3,449,083 6/1969 Pelick 210-31 C3,513,092 5/19 Matheane, Jr. 21031 C 3,600,306 8/1971 Tocci 210-31 CJOHN ADEE, Primary Examiner US. Cl. X.R. 210-198 C

